Oxygen plays a vital role in energy metabolism and immune defense in mammals, and it works within a narrow physiological range. The oxygen sensing mechanism enables mammalian cells to utilize oxygen effectively, defending against hypoxia as well as oxidative stress. Although the mechanism is poorly understood at the present, there is circumstantial evidence for a universal oxygen sensor inside cells. This research proposal aims to contribute to our knowledge on this topic by cloning and characterizing the human oxygen sensor. Biochemical approaches will be used to purify this sensory protein, a NAD(P)H oxidase on plasma membrane, and the cDNA will be cloned (Specific Aim 1). In vitro assays will he carried out to determine biochemical properties of the purified protein (Specific Aim 2) and in vivo studies at cellular and molecular levels will be performed on wild type oxygen sensor and its gain of function mutations, examining the downstream effect on hypoxia-regulated Epo gene expression (Specific Aim 3). A detailed understanding of the oxygen sensor will provide insights into the oxygen sensing pathway.